Detection of platelet-derived soluble P-selectin. To detect the shedding of platelet P-selectin into plasma, resting or thrombin-activated wild-type platelets were injected into P-selectin–deficient recipients. After 15 minutes mice were bled and plasma was isolated by centrifugation. (A) As revealed by ELISA, injection of resting platelets () did not induce significant changes in the level of soluble P-selectin compared with untreated P-selectin–deficient mice (□). Meanwhile, injection of thrombin-activated platelets (▪) generated a significant increase in soluble P-selectin in the plasma of the recipient mice (P < .0001). (B) A Western blot analysis with anti–P-selectin antibody showed the presence of an approximately 100-kD P-selectin fragment in mice injected with activated platelets (lane 4) but not with resting platelets (lane 3). Wild-type and P-selectin–deficient platelet lysates were used as reference (lane 1 and 2). In wild-type plasma from untreated animals, a small amount of P-selectin fragment was also detected (lane 5).